Core-shell structure drug delivery system loaded with oleuropein as well as preparation method and application of core-shell structure drug delivery system

Abstract

The invention relates to the technical field of nano-drug delivery systems, in particular to an oleuropein-loaded core-shell structure drug delivery system as well as a preparation method and application thereof. The invention relates to an oleuropein-loaded core-shell structure drug delivery system, which comprises an inner core and an outer shell, the inner core is an oleuropein nanoparticle formed by self-assembly of oleuropein; the oleuropein nanoparticles are cross-linked and modified with sodium tripolyphosphate and covalently connected with folic acid; the shell is formed from ferulic acid derived lignin. According to the scheme, efficient targeted delivery and collaborative treatment of the oleuropein in treatment of sepsis-related intestinal dysfunction are achieved, the technical problems that when the oleuropein is applied to treatment of sepsis, the drug targeting property is not high, the bioavailability is not ideal, and target site release cannot be achieved can be solved, and the application of the oleuropein to treatment of the sepsis is promoted. The invention opens up a new way for developing a novel efficient targeting drug delivery system, provides an innovative strategy for clinical conversion of natural products, and has ideal application and popularization values.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine delivery system technology, specifically to a core-shell structured drug delivery system loaded with oleuropein, its preparation method, and its application. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome triggered by infection, characterized by high morbidity and mortality, and currently lacks specific treatments. Research indicates that a vicious cycle between intestinal barrier dysfunction and mitochondrial damage plays a central role in the development and progression of sepsis. Intestinal ischemia-reperfusion injury can lead to increased mucosal barrier permeability, promoting endotoxin translocation and activating macrophages to produce large amounts of reactive oxygen species (ROS), thereby causing mitochondrial dysfunction. This positive feedback mechanism of "mitochondrial damage-inflammatory amplification" not only exacerbates tissue damage but also promotes systemic inflammatory responses, potentially leading to multiple organ dysfunction.

[0003] Oleuropein (OLE) is a natural polyphenolic compound derived from olive leaves. It has shown promising antioxidant, anti-inflammatory, and intestinal protective effects in various experimental models, particularly demonstrating therapeutic potential in animal models of sepsis. However, its clinical translation faces significant technical bottlenecks. First, the target of OLE is not yet fully understood, limiting a deeper understanding of its pharmacological mechanism and precise drug design. Second, its oral bioavailability is low, mainly due to degradation in the acidic environment of the stomach, the first-pass effect in the liver, and insufficient dose delivery efficiency.

[0004] Furthermore, existing oral delivery systems struggle to effectively accumulate OLE at sites of intestinal inflammation, particularly at the mitochondrial level, and lack responsiveness to oxidative stress and pH changes in the pathological microenvironment, thus affecting its release and efficacy at the target site. Therefore, there is an urgent need to develop a novel delivery system that overcomes these shortcomings and improves OLE targeting and bioavailability to enhance its application value in the treatment of sepsis-related intestinal injury. Summary of the Invention

[0005] The purpose of this invention is to provide a core-shell structured drug delivery system loaded with oleuropein to solve the technical problems of poor drug targeting, unsatisfactory bioavailability, and inability to achieve target site release when oleuropein (OLE) is used to treat sepsis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A core-shell structured drug delivery system loaded with oleuropein includes a core and a shell; the core is oleuropein nanoparticles formed by the self-assembly of oleuropein; the oleuropein nanoparticles are cross-linked with sodium tripolyphosphate and covalently linked with folic acid; the shell is formed from ferulic acid-derived lignin.

[0007] Furthermore, oleuropein nanoparticles are prepared by the following method: oleuropein is dispersed in dimethyl sulfoxide, and then the resulting mixed solution is added to water. After stirring, the mixture is centrifuged and the precipitate is collected. The precipitate is purified to obtain oleuropein nanoparticles. Preferably, 0.02-0.08 mmol of oleuropein monomer is dispersed in 1.0-4.0 mL of dimethyl sulfoxide; Preferably, 0.04 mmol of oleuropein monomer is dispersed in 2.5 mL of dimethyl sulfoxide.

[0008] Furthermore, sodium tripolyphosphate is crosslinked onto oleuropein nanoparticles in the following manner: oleuropein nanoparticles are dispersed in water, and then sodium tripolyphosphate solution is added to form a mixed solution of oleuropein nanoparticles and sodium tripolyphosphate; the pH value of the mixed solution of oleuropein nanoparticles and sodium tripolyphosphate is adjusted and stirred to obtain sodium tripolyphosphate crosslinked oleuropein nanoparticles. The mass ratio of oleuropein nanoparticles to sodium tripolyphosphate is 1:0.3-0.5; Preferably, the mass ratio of oleuropein nanoparticles to sodium tripolyphosphate is 1:0.4; Preferably, the solute mass concentration of the sodium tripolyphosphate solution is 0.8-1.8%; Preferably, oleuropein nanoparticles formed from 0.04 mmol of oleuropein monomer are dispersed in 1 mL of water.

[0009] Furthermore, folic acid is covalently linked to sodium tripolyphosphate-crosslinked oleuropein nanoparticles in the following manner: sodium tripolyphosphate-crosslinked oleuropein nanoparticles are dispersed in dimethyl sulfoxide, activated folic acid is added, and after reaction, folic acid and sodium tripolyphosphate-crosslinked oleuropein nanoparticles form a covalent link to obtain folic acid-modified nanoparticles; the mass ratio of sodium tripolyphosphate-crosslinked oleuropein nanoparticles to folic acid is 1:0.05-0.3. Preferably, the mass ratio of sodium tripolyphosphate cross-linked oleuropein nanoparticles to folic acid is 1:0.15; Preferably, oleuropein nanoparticles cross-linked with sodium tripolyphosphate, formed by reacting 0.04 mmol of oleuropein monomer, are dispersed in 1 mL of dimethyl sulfoxide at pH 7.4.

[0010] Furthermore, the outer shell is loaded onto the oleuropein nanoparticles in the following manner: folic acid-modified nanoparticles are dissolved in chloroform containing triethylamine, and a ferulic acid-derived lignin nanoparticle solution is added dropwise. After stirring, sonication, rotary evaporation to remove chloroform, and purification, a core-shell structured drug delivery system loaded with oleuropein is obtained. Preferably, 1-10g of folic acid-modified nanoparticles are dissolved in 0.5-2mL of chloroform containing 1-5μL of triethylamine, and 2-5mL of a 0.1-0.5% ferulic acid-derived lignin nanoparticle solution is added dropwise. Preferably, the volume of the ferulic acid-derived lignin nanoparticle solution is 3 mL, and the mass percentage of the solute is 0.3%; the folic acid-modified nanoparticles formed by reacting 0.04 mmol of oleuropein monomer are dispersed in 1 mL of chloroform containing 2.5 μL of triethylamine.

[0011] Furthermore, ferulic acid-derived lignin nanoparticles were prepared by the following method: A dimethyl sulfoxide solution of alkali lignin and a dimethyl sulfoxide solution of ferulic acid were prepared. After the ferulic acid in the dimethyl sulfoxide solution of ferulic acid was activated by the carboxyl group, the dimethyl sulfoxide solution of ferulic acid was added dropwise to the dimethyl sulfoxide solution of alkali lignin. After grafting reaction, purification, and freeze drying, ferulic acid-derived lignin powder was obtained. A dimethyl sulfoxide solution of ferulic acid-derived lignin powder was prepared. Under ice bath conditions, the dimethyl sulfoxide solution of ferulic acid-derived lignin powder was added dropwise to water while magnetic stirring was performed. After ultrasonic dispersion and purification, ferulic acid-derived lignin nanoparticles were obtained.

[0012] This technical solution also provides a method for preparing a core-shell structured drug delivery system loaded with oleuropein, comprising the following steps performed sequentially: S1: Olive bitter glycosides are dispersed in dimethyl sulfoxide, and the resulting mixed solution is added to water. After stirring, the mixture is centrifuged and the precipitate is collected. The precipitate is purified to obtain olive bitter glycoside nanoparticles. S2: Disperse oleuropein nanoparticles in water and then add sodium tripolyphosphate solution to form a mixed solution of oleuropein nanoparticles and sodium tripolyphosphate; adjust the pH value of the mixed solution of oleuropein nanoparticles and react with stirring to obtain oleuropein nanoparticles cross-linked with sodium tripolyphosphate. S3: Disperse sodium tripolyphosphate cross-linked oleuropein nanoparticles in dimethyl sulfoxide, add activated folic acid, and after reaction, folic acid and sodium tripolyphosphate cross-linked oleuropein nanoparticles form a covalent bond to obtain folic acid modified nanoparticles. S4: Folic acid-modified nanoparticles were dissolved in chloroform containing triethylamine, and a ferulic acid-derived lignin nanoparticle solution was added dropwise. After stirring, sonication, rotary evaporation to remove chloroform, and purification, a core-shell structured drug delivery system loaded with oleuropein was obtained.

[0013] Further, in S1, 0.02-0.08 mmol of oleuropein was dispersed in 1.0-4.0 mL of dimethyl sulfoxide, and then the resulting mixed solution was added to 6-25 mL of water. After stirring at 500-1500 rpm for 0.5-2 h, the mixture was centrifuged and the precipitate was collected. After purification, oleuropein nanoparticles were obtained. In S2, the mass ratio of oleuropein nanoparticles to sodium tripolyphosphate is 1:0.3-0.5; the pH of the oleuropein nanoparticle-sodium tripolyphosphate mixed solution is adjusted to 4.5-5.5 and stirred at 200-800 rpm for 4-8 hours to obtain sodium tripolyphosphate cross-linked oleuropein nanoparticles.

[0014] Furthermore, in S3, the ratio of sodium tripolyphosphate-crosslinked oleuropein nanoparticles to folic acid is 1:0.05-0.3; In S4, 1-10 g of folic acid-modified nanoparticles were dissolved in 0.5-2 mL of chloroform containing 1-5 μL of triethylamine. 2-5 mL of a 0.1-0.5 wt% ferulic acid-derived lignin nanoparticle solution was added dropwise. After stirring in an ice bath at 8000-12000 rpm for 3-10 min, sonicating at 100-500 W for 1-5 min, removing chloroform by rotary evaporation, and purification, a core-shell structured drug delivery system loaded with oleuropein was obtained.

[0015] This technical solution also provides the application of a core-shell structured drug delivery system loaded with oleuropein in the preparation of drugs for treating sepsis, which is used to improve the pathological damage to intestinal tissues caused by sepsis.

[0016] In summary, the technical principle of this solution is as follows: This invention provides a core-shell structured drug delivery system loaded with oleuropein (OLE), aiming to address the technical problems of poor drug targeting, low bioavailability, and inability to achieve target site release in the treatment of sepsis using existing OLE technologies. The drug delivery system consists of a core and a shell: the core is oleuropein nanoparticles formed through self-assembly, and its stability is enhanced by cross-linking with sodium tripolyphosphate (TPP); it is covalently linked to folic acid (FA) to achieve active recognition of folic acid receptors highly expressed at inflammatory sites; the shell is formed from ferulic acid-derived lignin nanoparticles (FALNP), endowing the system with pH / enzyme-responsive controlled-release properties. Specifically, oleuropein is first dispersed in dimethyl sulfoxide (DMSO), then the mixture is added to water, stirred, centrifuged, and the precipitate is collected and purified to obtain oleuropein nanoparticles. Next, the oleuropein nanoparticles are cross-linked using sodium tripolyphosphate, the pH of the mixture is adjusted, and the reaction is stirred at room temperature to form stable nanoparticles. Subsequently, activated folic acid was covalently linked to oleuropein nanoparticles cross-linked with sodium tripolyphosphate to prepare folic acid-modified nanoparticles. Finally, the folic acid-modified nanoparticles were dissolved in chloroform containing triethylamine, and a ferulic acid-derived lignin nanoparticle solution was added dropwise. After stirring, sonication, rotary evaporation to remove chloroform, and purification, a core-shell structured drug delivery system loaded with oleuropein was finally obtained.

[0017] This approach utilizes a core-shell drug delivery system, FALNP@FTO, to achieve highly efficient targeted delivery and synergistic therapy of oleuropein (OLE) in the treatment of sepsis-related intestinal dysfunction. The core principle is as follows: oleuropein self-assembles into nanoparticles (OLE-NP) through hydrophobic interactions and hydrogen bonds. After TPP cross-linking to enhance stability, it actively recognizes folate receptors highly expressed at inflammatory sites using folic acid (FA) targeting modification. Combined with the pH / enzyme-responsive controlled-release properties of the lipid shell (FALNP), precise accumulation and intelligent release of the drug at the lesion site are achieved. This system exerts its therapeutic effect through a triple synergistic mechanism: ① oleuropein inhibits the NF-κB pathway, reducing cytokine storms, while the phenolic hydroxyl groups directly scavenge free radicals to alleviate oxidative stress; ② it disrupts bacterial membrane integrity to exert antibacterial effects; ③ it regulates macrophage polarization and T cell subset balance to maintain immune homeostasis. Its structural advantages include: a clear division of labor between the core layer (drug loading) and the shell layer (controlled release barrier); dual enrichment through FA targeting and EPR effect (enhanced permeability and retention effect); and synergistic enhancement of stability through cross-linking network and lipid encapsulation. Furthermore, it significantly reduces systemic toxicity by minimizing off-target effects, providing an innovative strategy for the clinical translation of natural products.

[0018] The beneficial effects of this technical solution are as follows: (1) Highly efficient targeted delivery: The active recognition of folic acid receptors highly expressed at the site of inflammation was achieved through folic acid modification. Combined with the EPR effect, the accumulation efficiency of nanoparticles at the site of inflammation was significantly improved compared with traditional carriers.

[0019] (2) Enhanced stability and controlled release performance: The use of sodium tripolyphosphate cross-linking enhances the stability of the nanoparticles, effectively reducing the drug leakage rate within 24 hours. At the same time, the ferulic acid-derived lignin shell provides a pH / enzyme-responsive intelligent release mechanism, effectively improving the drug release rate at the lesion site and ensuring effective drug release.

[0020] (3) Synergistic treatment by multiple mechanisms: The natural ingredient oleuropein reduces the inflammatory storm (such as the decrease in TNF-α / IL-6 levels) by inhibiting the NF-κB pathway, directly scavenges free radicals to relieve oxidative stress, and destroys bacterial membranes to exert multiple effects of anti-inflammatory, antioxidant and antibacterial, significantly improving the survival rate in CLP mouse models.

[0021] (4) Safety and biodegradability: The optimized nanoparticle design effectively reduces hepatotoxicity and nephrotoxicity. In addition, all materials are biodegradable, ensuring good biocompatibility and safety.

[0022] (5) Simple and efficient preparation process: The entire preparation process is simple and quick, with short time consumption, suitable for large-scale production, and provides an economical and effective solution for the clinical translation of natural products.

[0023] In summary, this invention not only solves the key technical bottlenecks encountered by OLE in the treatment of sepsis, but also demonstrates its great potential in improving the pathological damage to intestinal tissues caused by sepsis, opening up new avenues for the development of novel and efficient targeted drug delivery systems. Attached Figure Description

[0024] Figure 1 The diagram shows the structures and preparation process of the oleuropein nanoparticles OLE-NPs, TPP-crosslinked nanoparticles TO-NPs, folic acid-modified nanoparticles FTO-NPs, and core-shell structured nanoparticles FALNP@FTO from Example 1.

[0025] Figure 2 This is a particle size distribution characteristic diagram of the FALNP@FTO nanoparticles in Example 1.

[0026] Figure 3 This is a comparison graph of particle size and zeta potential for OLE-NPs, FTO-NPs, and FALNP@FTO nanoparticles in Example 1.

[0027] Figure 4This is a statistical chart showing the dynamic changes in nanoparticle size and polydispersity index (PDI) from 0 to 7 days in Example 1.

[0028] Figure 5 The effects of different drugs or nanoparticles (OLE, OLE-NPs, FTO-NPs, FALNP@FTO) on cell survival in Example 2.

[0029] Figure 6 The results of the targeting effects of different drugs or nanoparticles (OLE, OLE-NPs, FTO-NPs, FALNP@FTO) on cellular mitochondria in Example 2 are analyzed.

[0030] Figure 7 The results of the analysis of the effects of different drugs or nanoparticles (OLE, OLE-NPs, FTO-NPs, FALNP@FTO) on the level of reactive oxygen species (mito-ROS) in cellular mitochondria in Example 2 are as follows.

[0031] Figure 8 The results of the experiment evaluating the fluorescence distribution and targeting of different drugs or nanoparticles (OLE, OLE-NPs, FTO-NPs, FALNP@FTO) in mice in Example 2 are presented.

[0032] Figure 9 This is the result of a study on the effects of different drugs or nanoparticles (OLE, OLE-NPs, FTO-NPs, FALNP@FTO) on the infiltration of F4 / 80 and MPO positive cells in mouse tissues and the drug targeting of Example 3.

[0033] Figure 10 This is the result of the study on the effects of different drugs or nanoparticles (OLE, OLE-NPs, FTO-NPs, FALNP@FTO) on the histopathological morphology and histological scores of mouse tissues in Example 3.

[0034] Figure 11 The particle size distribution of OLE-NPs prepared using the ethanol-water system is shown in Comparative Example 1.

[0035] Figure 12 The image shows an electron microscope image of OLE-NPs prepared at a stirring rate of 6000 rpm, which is Comparative Example 2.

[0036] Figure 13 The images are electron microscope images of OLE-NPs from Example 1 and Comparative Example 2, obtained by stirring at 1000 rpm. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0038] Example 1: Preparation of ROS / pH Dual-Response Core-Shell Nanoparticle Drug Delivery System FALNP@FTO (1) Composition of FALNP@FTO nanoparticles The core components of the nanoparticles consist of two parts: oleuropein nanoparticles (OLE-NP core) and FALNP shell.

[0039] OLE-NP core: Based on oleuropein (OLE, CAS No.: 32619-42-4), its amphiphilic molecular structure (containing hydrophilic groups such as hydroxyl and ester groups and hydrophobic carbon rings) self-assembles into nanoparticles through antisolvent precipitation. Its phenolic hydroxyl groups serve as ROS response sites, and the particles can be dissociated through redox reactions.

[0040] FALNP shell: composed of ferulic acid-derived lignin nanoparticles, prepared by in vitro free radical polymerization, has the characteristics of strong acid stability and dissociation under alkaline conditions, and has the advantages of low toxicity, renewability and antioxidant properties, and can be used as an intestinal pH-responsive shell to optimize drug delivery.

[0041] The two combine to form a core-shell structure. The OLE-NP core loads the therapeutic active ingredient and achieves ROS-responsive release, while the FALNP shell protects the drug from gastric acid degradation through pH response, synergistically enhancing the oral bioavailability and targeted therapeutic effect of oleuropein.

[0042] (2) Preparation method of FALNP@FTO nanoparticles (2.1) Preparation of oleuropein nanoparticles (OLE-NP): 0.04 mmol (optional range: 0.02-0.08 mmol) of oleuropein monomer (OLE) was dispersed in 2.5 mL of dimethyl sulfoxide (DMSO, optional range: 1.0-4.0 mL). The solution formed by mixing OLE and DMSO was added to 12.5 mL (optional range: 6-25 mL) of deionized water and stirred at 1000 rpm (optional range: 500-1500 rpm) for 1 h (optional range: 0.5-2 h). Then, the mixture was centrifuged at 1000 rpm for 5 min and the precipitate was collected. The centrifugation, discarding of supernatant, and collection of precipitate were repeated 3 times to wash the nanoparticles to remove unreacted free OLE and other impurities. The final precipitate was dialyzed with water (dialysis bag with Mw cutoff value of 6000–8000 g / mol) for 72 h to remove DMSO, and then the precipitate was dispersed in water and stored at 4 °C. Oleuropein nanoparticles (OLE-NPs) were obtained by conventional freeze-drying.

[0043] (2.2) TPP crosslinking modification: The lyophilized OLE-NP was dispersed in 1 mL of deionized water (approximately 21.62 mg / mL), and ultrasonic treatment was performed to ensure uniform dispersion. The ultrasonic treatment parameters were as follows: ultrasonic power 100-300 W (preferably 200 W); ultrasonic duration 3-8 min (preferably 5 min); ultrasonic mode was intermittent, with 30 s of operation followed by a 10 s pause to avoid local overheating and degradation of the OLE structure; temperature control was performed in an ice-water bath (0-4℃) to reduce the impact of ultrasonic thermal effects on the stability of the nanoparticles. Then, sodium tripolyphosphate solution (TPP, solute mass concentration 0.8-1.8%, preferably 1%) is slowly added, and the mass ratio of OLE-NPs:TPP is 1:0.3-0.5, preferably 1:0.4 in this scheme. The pH of the mixed solution is adjusted to 4.5-5.5, and TPP is combined with the hydroxyl groups on the surface of OLE-NPs through esterification reaction. The mixture is stirred magnetically at 1000 rpm (optional range 200-800 rpm) for 5 h (optional range 4-8 h), and purified by centrifugation to obtain TPP cross-linked nanoparticles (TO-NPs, sodium tripolyphosphate cross-linked oleuropein nanoparticles).

[0044] (2.3) Disperse TO-NPs in 1 mL of DMSO solution (pH 7.4, approximately 30.27 mg / mL; i.e., disperse all TO-NPs obtained in the previous steps in DMSO), and add activated folic acid FA (pre-activated carboxyl groups using conventional EDC / NHS method). The mass ratio of TO-NPs to FA is 1:0.05-0.3, preferably 1:0.15 in this scheme. The carboxyl groups of FA are covalently linked to the amino groups on the surface of TO-NPs through a condensation reaction; after reacting at room temperature for 12 h, dialyze using a 1000D dialysis bag (molecular weight cutoff 1000 g / mol) to remove unreacted FA, and freeze-dry to obtain folic acid-modified nanoparticles (FTO-NPs).

[0045] The activation of folic acid (FA) is a conventional method in existing technology. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and EDC (N-hydroxysuccinimide) are a classic cross-linking reagent combination widely used in biochemistry and materials science. By pre-activating the carboxyl group of folic acid with EDC / NHS, NHS is attached to the carboxyl group, forming an FA-NHS ester, thus activating the carboxyl group. In this way, the activated carboxyl group (-COOH) promotes its covalent coupling reaction with the primary amino group (-NH2). More specifically, a PBS buffer at pH 7.4 is used as the reaction environment to maintain the same pH as the solution formed by dispersing TO-NPs in DMSO solution in this step, avoiding pH differences that could affect subsequent covalent bonding. The feed ratio was FA:EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide):NHS (N-hydroxysuccinimide) = 1:1.5:1.5 (adapted to the conventional ratio for carboxyl-amino condensation, avoiding side reactions caused by excessive EDC / NHS). The concentration of FA was 2 mM. After a conventional reaction at room temperature (20-25℃) for 1.5 h, followed by conventional purification, activated folic acid (FA-NHS ester) was obtained. The entire process was carried out in the dark before subsequent covalent bonding with TO-NPs.

[0046] (2.4) Core-shell structure assembly: The emulsion solvent evaporation method was used to load nanoparticles (FTO-NPs). All FTO-NPs obtained in the above process (optional range 1-10 g) were dissolved in chloroform (1 mL, containing 2.5 μL triethylamine; optional range 0.5-2 mL, 1-5 μL). FALNP dispersion (0.3 wt%, 3 mL, ferulic acid-derived lignin nanoparticles; optional range 0.1-0.5 wt%, 2-5 mL) was gradually added dropwise to the solution. The system was then stirred in an ice-water bath (10,000 rpm, optional range 8000-12000 rpm) for 5 min (optional range 3-10 min) to form a preliminary emulsion. The mixture was ultrasonically treated at 300 W (100-500 W) for 3 min (optional range 1-5 min) to obtain a stable nanoemulsion. The chloroform was then removed using a rotary evaporator to obtain FALNP@FTO. The FALNP@FTO nanoparticle dispersion was lyophilized and stored in the dark.

[0047] FALNP is prepared by the following method: Ferulic acid-derived lignin is a functionalized lignin derivative formed by the covalent grafting reaction of ferulic acid (FA) with the active groups (such as hydroxyl and carboxyl groups) of lignin molecules. It is not a single substance; its core characteristic is the retention of the aromatic ring skeleton of lignin and the phenolic hydroxyl / carboxyl structure of ferulic acid. It possesses both pH responsiveness (the carboxyl group of ferulic acid protonates under acidic conditions and dissociates under alkaline conditions) and antioxidant activity (the phenolic hydroxyl groups of both synergistically scavenge ROS). Based on existing technology and the application scenario of this invention (intestinal pH-responsive drug delivery), the ferulic acid-derived lignin used in this scheme is specifically ferulic acid-grafted alkali lignin. The grafting site is the hydroxyl group exposed after the β-O-4 ether bond of lignin is broken, which is then linked to the carboxyl group of ferulic acid via an ester bond to form the derivative. Its structure is verified by infrared spectroscopy (IR): at 1720 cm⁻¹... -1 A characteristic absorption peak for ester bonds appears at 3400 cm⁻¹. -1 The intensity of the hydroxyl absorption peak decreased (indicating that the grafting reaction occurred), and the UV spectrum showed a characteristic absorption peak of ferulic acid at 320 nm (distinguishing it from the original lignin).

[0048] Specifically, the preparation method is as follows: The reagents used included: alkali lignin (industrial grade, purity ≥90%, UPM): as the parent compound, providing the aromatic ring skeleton and grafting sites; ferulic acid (analytical grade, CAS No.: 1135-24-6): providing carboxyl and phenolic hydroxyl groups, imparting pH responsiveness and targeted auxiliary functions; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS): as carboxyl activators, promoting the ester bond connection between ferulic acid and lignin; dimethyl sulfoxide (DMSO), deionized water, and triethylamine (TEA).

[0049] The preparation process consists of two steps: ferulic acid-derived lignin synthesis and nanoparticle preparation.

[0050] The synthesis (grafting reaction) of ferulic acid-derived lignin is as follows: Lignin pretreatment: Take 1.0 g of alkali lignin, dissolve it in 20 mL of DMSO, add 0.5 mL of TEA to adjust the pH to 8.0, stir at 500 rpm for 30 min, and sonicate (200 W, 5 min) to remove agglomerates to obtain lignin-DMSO solution.

[0051] Ferulic acid activation: Take 0.3g ferulic acid (about 30% of the lignin quality, the optimal ratio for grafting), dissolve it in 10 mL DMSO, add 0.24g EDC and 0.14g NHS (FA:EDC:NHS molar ratio of 1:1.2:1.2), stir at room temperature in the dark for 1 h to activate the carboxyl group of ferulic acid.

[0052] Grafting reaction: The activated ferulic acid solution was slowly added dropwise to the lignin-DMSO solution and stirred at 30℃ for 8 hours (the reaction temperature and time were optimized by orthogonal experiments to ensure that the grafting rate was ≥25%).

[0053] Purification: After the reaction was completed, the mixture was added dropwise to 50 mL of deionized water (antisolvent precipitation), centrifuged at 10000 rpm for 10 min, and the precipitate was collected; dialyzed with deionized water (molecular weight cutoff 8000 g / mol) for 72 h, with the solution changed 3 times a day to remove unreacted ferulic acid and EDC / NHS.

[0054] Drying: Ferulic acid-derived lignin powder was obtained after freeze drying. The grafting rate was determined by ultraviolet spectrophotometry (calculated using the ferulic acid standard curve, with the grafting rate controlled at 25%-30% to ensure a balance between pH responsiveness and solubility).

[0055] The specific process for preparing ferulic acid-derived lignin nanoparticles (FALNP) (antisolvent precipitation-ultrasound-assisted method) is as follows: Solution preparation: Take 0.15g of ferulic acid-derived lignin powder, dissolve it in 5mL of DMSO, stir at 500rpm for 1h to form a homogeneous derivative solution (concentration 30mg / mL).

[0056] Antisolvent precipitation: Under ice bath conditions, the above solution was slowly added dropwise to 45 mL of deionized water at a rate of 10 mL / h (DMSO:water volume ratio of 1:9 to avoid particle agglomeration), while stirring magnetically at 800 rpm.

[0057] Ultrasonic dispersion: After the addition is complete, ultrasonic treatment is performed (300W, 3min, intermittent: 30s working, 10s pause) to further refine the particles and avoid ester bond hydrolysis caused by thermal effects.

[0058] Purification and storage: Centrifuge at 10,000 rpm for 20 min to remove large particle size aggregates; collect the supernatant, filter through a 0.22 μm filter membrane for sterilization, and obtain FALNP dispersion (concentration controlled at 0.3 wt%). Store at 4℃ in the dark; particle size change ≤10% within 7 days (verified by a nanoparticle size analyzer).

[0059] (2.5) Purification and preservation: Add 5-10 mL of deionized water to the concentrated (or semi-solid) product after rotary evaporation, and perform magnetic stirring (500 rpm, 5 min) + ultrasonic dispersion (300 W, 2 min, intermittent) to uniformly disperse FALNP@FTO nanoparticles in the aqueous phase, while simultaneously dissolving residual soluble impurities. Place the dispersion in a centrifuge tube, centrifuge at 10000 rpm for 20 min, and discard the supernatant (containing impurities). Add 5 mL of deionized water to the precipitate again, and repeat the "dispersion-centrifugation" operation twice to ensure complete removal of impurities. Collect the final centrifuged precipitate, then freeze-dry and store at 4°C in the dark. Washing and centrifugation remove free FALNP, triethylamine, and other impurities, ensuring FALNP@FTO purity ≥95% and avoiding the adverse effects of impurities. The washing process removes excess FALNP adsorbed on the surface, preventing particle agglomeration after freeze-drying and ensuring the particle size remains within the 100-1000 nm range. This washing process removes potentially toxic impurities such as triethylamine, further reducing hepatotoxicity and nephrotoxicity.

[0060] For detailed structural diagrams and preparation process diagrams of oleuropein nanoparticles (OLE-NPs), TPP-crosslinked nanoparticles (TO-NPs), folic acid-modified nanoparticles (FTO-NPs), and core-shell structured nanoparticles (FALNP@FTO), please refer to [link to relevant documentation]. Figure 1 .

[0061] (3) Characterization of nanoparticles Experimental measurements revealed that the FALNP@FTO nanoparticles involved in this invention exhibit specific particle size distribution characteristics. The particle size of the FALNP@FTO nanoparticles is mainly distributed in the 100-1000 nm range. This distribution indicates that a high proportion of particles in this size range are present in the nanoparticle system. Furthermore, the particle size distribution curve is narrow and sharp, indicating good uniformity in particle size distribution and relatively similar particle sizes, which is beneficial for achieving more stable in vivo distribution during drug delivery. See the particle size distribution diagram for details. Figure 2 .

[0062] The particle size and zeta potential of three types of nanoparticles—OLE-NPs, FTO-NPs, and FALNP@FTO—were measured. Figure 3 As shown, there are significant differences in particle size among the three types of nanoparticles. The particle size of FALNP@FTO nanoparticles is significantly larger than that of OLE-NPs, and the difference is highly statistically significant. The zeta potential values ​​of the three types of nanoparticles are all at low levels, and there is no statistically significant difference among them.

[0063] The particle size (nm) and polydispersity index (PDI) of the relevant nanoparticle system were monitored at different time points, spanning 0-7 days. Figure 4 It can be seen that the nanoparticle size remained within a certain range throughout the experiment. Initially, the particle size was approximately 162 nm. On the first day, the particle size decreased slightly, then gradually increased from the second day, reaching a relative peak on the fourth day, after which it fluctuated. The PDI value changed synchronously during the experiment, and its fluctuations reflected changes in the dispersion state of the nanoparticles.

[0064] Example 2: In vitro experiment (1) Cell viability assay Raw264.7 macrophages (density 1×10⁻⁶) 4 Cells (per well) were seeded into 96-well plates. Different concentrations of OLE, OLE-NPs, FTO-NPs, and FALNP@FTO were added to the experimental groups. The negative control group consisted of untreated cells, the blank control group consisted of cell-free culture medium, and the positive control group consisted of cisplatin (concentration adjusted according to cell type). 24-48 hours after drug intervention, 10 μL CCK-8 reagent was added to each well, and the cells were incubated at 37°C in the dark for 1-4 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability was calculated using the formula: (Experimental group OD - Blank OD) / (Negative control OD - Blank OD) × 100%.

[0065] For detailed experimental data, please refer to Figure 5 Under low concentration (0-150 μg / mL, based on OLE) treatment conditions, cell viability in all treatment groups was close to or exceeded 100%, indicating no significant toxicity at low concentrations; in fact, some groups (e.g., FTO-NPs at 50 μg / mL, 112%) may have slightly promoted cell proliferation. Under high concentration (200-250 μg / mL) treatment conditions, cell viability in all groups remained at a high level, indicating that encapsulation reduced drug toxicity and enhanced biocompatibility.

[0066] (2) Verification of macrophage mitochondrial targeting Raw264.7 cell lines were revived and cultured to the logarithmic growth phase. Cells were then plated at an appropriate density until adherence and confluence reached 70%-80%. Subsequently, cells were grouped and treated with drugs, into OLE, OLE-NPs, FTO-NPs, and FALNP@FTO groups. The original culture medium was discarded and the cells were washed, then incubated with serum-free medium containing the corresponding reagents at equal concentrations (10 μg / mL, based on OLE). Cells were then labeled using conventional techniques with Mitotracker (mitochondrial labeling), DAPI (nuclear labeling), and DiD (target drug fluorescent labeling). Cell images were then acquired using a confocal microscope with corresponding channels for DAPI, Mitotracker, and DiD, and with standardized laser intensity and other parameters. Finally, fluorescence intensity and localization were analyzed using software such as ImageJ, and the images were processed to present the experimental results. The statistical indicator was "cellular DID level (%)", specifically the relative intensity percentage of intracellular DiD fluorescence signal.

[0067] For detailed experimental results, please refer to Figure 6 The OLE group showed weak and dispersed green fluorescence, indicating that the amount of free drug (OLE) entering the cells was small, or that there was no significant aggregation within the cells. The FALNP@FTO group showed enhanced green fluorescence and a certain aggregation trend, indicating that the nanoparticles had improved targeted uptake ability by cells, possibly through receptor-mediated endocytosis. The FTO-NPs group showed the strongest green fluorescence, concentrated in specific regions of the cell (such as the cytoplasm), and some cells showed obvious particle aggregation.

[0068] (3) Detection of the effect of FALNP@FTO on cellular ROS levels LPS-induced Raw264.7 macrophages were seeded in 6-well plates (density 1×10⁻⁶). 6 Cells / wells were used to treat experimental groups with OLE, OLE-NPs, FTO-NPs, and FALNP@FTO (10 μg / mL OLE equivalent) for 12 h, respectively. The positive control group received antimycin A (10 μM, to induce mtROS). After removing the culture medium, pre-warmed Hanks balanced salt solution (HBSS buffer) containing 5 μM MitoSOX Red was added, and the cells were incubated in the dark for 10 min. Unbound probes were removed by washing three times with PBS. Mitochondrial ROS levels were detected using confocal microscopy and DAPI co-localization. See detailed experimental results below. Figure 7Compared to the OLE group, the levels of mitochondrial reactive oxygen species (mito-ROS) in the OLE-NPs, FTO-NPs, and FALNP@FTO groups were significantly decreased, especially in the FTO-NPs and FALNP@FTO groups. The results are expressed as "Relative mito-ROS level (%)", representing the relative fluorescence intensity percentage of reactive oxygen species (mito-ROS) in mitochondria. MitoSOX Red is a mitochondrial-specific ROS fluorescent probe (emitting red fluorescence only after being oxidized by ROS in mitochondria).

[0069] Example 3: In vivo experiment (1) Validation of gut targeting Twelve healthy Balb / c mice were selected to construct a CLP model (cecal ligation and perforation model, an animal model widely used in sepsis research). The mice were randomly divided into four groups. OLE (free drug), OLE-NPs (nanoparticle drug), FTO-NPs (nanoparticles targeting macrophage mitochondria), and FALNP@FTO (nanoparticles targeting intestinal macrophage mitochondria) were labeled with Cy5.5, respectively. Each group was administered the corresponding formulation via gavage at the same dose. More specifically, each group received a single dose of 10 mg / kg of OLE (100 μL / mouse). "Equal dose" refers to the same dose based on the actual content of oleuropein (OLE) to ensure consistent dosage of the active ingredient across groups and comparability of experimental results. Mice were anesthetized at 2, 4, and 8 hours after gavage and placed in an in vivo imaging system. Whole-body fluorescence images of the mice were acquired, focusing on the fluorescence distribution and intensity of the colon and liver. The fluorescence intensity of the colon and liver was quantitatively analyzed using imaging system software. Statistical analysis was performed on the data to compare differences between groups. For detailed experimental results, please refer to Figure 8 .

[0070] At 2 hours, Cy5.5 fluorescence signals were observed in all groups of mice. The FALNP@FTO group showed relatively concentrated and stronger fluorescence signals in the abdomen (corresponding to organs such as the colon), while the OLE and OLE-NPs groups showed diffuse and weaker fluorescence signals. At 4 hours, the abdominal fluorescence signal in the FALNP@FTO group further increased, with clearer colonic localization. Although the FTO-NPs group showed enrichment, its intensity and concentration were not as high as the FALNP@FTO group. The fluorescence signals in the OLE and OLE-NPs groups began to weaken, but the distribution remained relatively scattered. At 8 hours, the colonic fluorescence signal in the FALNP@FTO group was significantly stronger than that in the other groups, showing obvious targeted enrichment characteristics. The fluorescence signals in the other groups (such as the OLE and OLE-NPs groups) further weakened, and the intensity of the FTO-NPs group was also lower than that of the FALNP@FTO group, highlighting the colonic enrichment advantage of FALNP@FTO. Quantitative analysis of organ fluorescence intensity showed that the colon fluorescence intensity in the FALNP@FTO group was significantly higher than that in other groups, indicating superior targeting. The liver fluorescence signal in the OLE-NPs group was significantly stronger than that in other groups. There were no significant differences in liver fluorescence intensity among the other groups, suggesting that OLE-NPs treatment had a greater impact on liver targeting. In summary, FALNP@FTO exhibits superior targeting enrichment ability in the colon compared to other groups, with the effect becoming more significant over time. The lack of significant differences in liver fluorescence intensity among the groups confirms that this formulation possesses superior targeting properties for the colon.

[0071] (2) Validation of intestinal macrophage targeting The regulatory effects of different nano-formulations on macrophage infiltration, neutrophil activity, and targeting were systematically evaluated using immunofluorescence staining combined with quantitative analysis. Detailed experimental results can be found in [link to results]. Figure 9The results showed that in F4 / 80-labeled macrophages, the percentage of positive cells in the FALNPs@FTO group (<10%) and the FTO-NPs group (around 10%) was significantly lower than that in the OLE-NPs group (around 20%) and the OLE group (around 30%), indicating that FALNPs@FTO can effectively inhibit macrophage infiltration. In MPO-labeled neutrophils, the percentage of positive cells in the FALNPs@FTO and FTO-NPs groups was between 10-20%, the percentage in the OLE-NPs group was between 20-30%, and the percentage in the OLE group was approximately 30%. FALNPs@FTO and FTO-NPs showed more ideal effects, further reducing inflammatory responses, immune cell infiltration, and oxidative stress levels. DiD-labeled nanoparticles showed that the FALNPs@FTO group had the highest targeting positivity rate (20-30%), significantly better than OLE-NPs (around 10%) and OLE (around 10%), confirming that its core-shell structure significantly enhances the targeting and retention capacity of OLE tissue. In summary, FALNPs@FTO provides a new strategy for long-acting therapy with its precise delivery characteristics, and it does not induce abnormal neutrophil activation, demonstrating good safety.

[0072] (3) Therapeutic effects of nanoparticles Mouse colon tissue was collected, fixed in formaldehyde solution, dehydrated, cleared, and then embedded in paraffin to form a paraffin block. The paraffin block was cut into 3-5 μm thin sections using a microtome and mounted on glass slides. Hematoxylin staining, hydrochloric acid-alcohol differentiation, and eosin staining were then performed sequentially, resulting in blue nuclei and pink cytoplasm and extracellular matrix. Finally, the stained sections were observed and photographed under a light microscope at an appropriate field of view.

[0073] For detailed experimental results, please refer to Figure 10 The experimental results showed that the OLE group exhibited significant intestinal tissue damage, characterized by severe colonic mucosal defects, loss of normal intestinal epithelial morphology in some areas, reduced glandular numbers, mucosal swelling, and a large accumulation of inflammatory cells in both the mucosal and submucosa. In contrast, the OLE-NPs group showed significantly reduced intestinal epithelial structural damage, increased glandular numbers, and decreased inflammatory infiltration, suggesting that OLE effectively alleviates CLP-induced intestinal damage. The FTO-NPs and FALNP@FTO groups showed intact intestinal mucosal structures, tightly packed glands, and minimal inflammatory cell infiltration, with no obvious pathological damage. This part of the experimental results confirms that FALNP@FTO can effectively improve the pathological damage of intestinal tissue in CLP mice.

[0074] Comparative Example 1: Effect of solvent selection on the particle size of oleuropein nanoparticles (OLE-NP) This technical solution yielded oleuropein nanoparticles (OLE-NP). The key technical point lies in using DMSO as a dissolver and adding a mixture of OLE and DMSO to water, thereby allowing OLE-NP to self-assemble. Prior to determining the above preparation method, the inventors conducted extensive research on OLE self-assembly processes. Because OLE molecules simultaneously contain hydrophilic hydroxyl / ester groups and hydrophobic benzene rings, the use of antisolvent precipitation methods easily leads to uneven molecular packing, ultimately forming irregular polydisperse particles, posing a significant challenge to the development of the nanoparticles described in this solution.

[0075] Before using the preparation process of Example 1, the inventors prepared OLE-NPs in the following manner: Compared with the optimal implementation method of Example 1, the main difference in the preparation of OLE-NPs lies in the solvent (DMSO is replaced with anhydrous ethanol), while the other parameters are basically the same as in Example 1. Specifically, 0.04 mmol of OLE monomer was accurately weighed (calculated based on the molar mass of OLE 540.5 g / mol, the corresponding mass is 0.04 mmol × 540.5 g / mol = 0.02162 g, or 21.62 mg), and dispersed in 2.5 mL of anhydrous ethanol. Magnetic stirring was used to ensure that the OLE was dissolved as completely as possible. At room temperature (20-25℃), the above OLE-ethanol mixture was slowly added dropwise to 12.5 mL of deionized water at a rate of 1 mL / min, while simultaneously stirring magnetically at 1000 rpm for 1 h to form an OLE suspension system. Subsequently, the mixture was washed, dialyzed, purified, and freeze-dried as in Example 1 to obtain powdered oleuropein nanoparticles OLE-NPs.

[0076] Characterization of the OLE-NPs prepared by the above method revealed that the final particle size was too large, deviating significantly from the expected particle size and failing to meet application requirements (see particle size distribution diagram for details). Figure 11 The particle size distribution is basically in the range of 1000-10000 nm. Based on experimental observations, the inventors analyzed the reasons: OLE molecules may have undergone heterogeneous nucleation in ethanol, forming large particles (particle size > 500 nm), which are prone to coarsening (PDI > 0.3). Furthermore, ethanol, as a proton solvent, may catalyze the hydrolysis of ester bonds (acid-catalyzed pathway), leading to a certain degree of degradation of OLE.

[0077] In Example 1, DMSO was used instead of ethanol to ensure that OLE formed a homogeneous molecular dispersion before antisolvent precipitation. The DMSO-water system promotes uniform nucleation, resulting in small-diameter particles. DMSO does not hydrolyze the ester bonds of OLE. DMSO ensures that OLE molecules do not undergo heterogeneous nucleation, achieving nanoparticle homogenization and protecting the structural integrity of OLE. In Example 1, a DMSO-water system (solvent ratio DMSO:water = 1:5) was selected as the OLE solvent to re-prepare OLE-NPs, ultimately controlling their particle size within the ideal range.

[0078] Comparative Example 2: Effect of stirring rate on the uniformity of OLE-NP Studies have found that the DMSO-water system is crucial for maintaining a small OLE-NP particle size; however, controlling only this factor does not result in uniform particle size in the prepared OLE-NP nanoparticles. Through extensive experimentation, the inventors discovered that controlling the rotational speed is critical for controlling the nanoparticle size.

[0079] OLE-NPs were prepared as follows: 0.04 mmol of oleuropein monomer (OLE) was dispersed in 2.5 mL of dimethyl sulfoxide (DMSO). The solution formed by mixing OLE and DMSO was added to 12.5 mL of deionized water and stirred at 6000 rpm for 1 h. The remaining steps were performed according to the optimal method of Example 1. The OLE-NPs prepared by this method were observed by electron microscopy, see [link to example]. Figure 12 In this preparation process, the stirring rate was 6000 rpm. Excessive stirring speed caused shear forces that disrupted the structure of the self-assembled particles, while insufficient stirring speed led to particle aggregation and excessively large particle sizes. After repeated experiments, the stirring rate was finally controlled at 1000 rpm, resulting in uniform and stable OLE-NPs with particle sizes within the ideal range (see...). Figure 13 (Prepared according to the method of Example 1).

[0080] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A core-shell structured drug delivery system loaded with oleuropein, characterized in that: It comprises a core and a shell; the core is oleuropein nanoparticles formed by the self-assembly of oleuropein; the oleuropein nanoparticles are cross-linked with sodium tripolyphosphate and covalently linked with folic acid; the shell is formed from ferulic acid-derived lignin.

2. The core-shell structured drug delivery system loaded with oleuropein according to claim 1, characterized in that: Olive bitter glycoside nanoparticles were prepared by the following method: olive bitter glycoside was dispersed in dimethyl sulfoxide, and then the resulting mixed solution was added to water. After stirring, the mixture was centrifuged and the precipitate was collected. After purification, olive bitter glycoside nanoparticles were obtained. Preferably, 0.02-0.08 mmol of oleuropein monomer is dispersed in 1.0-4.0 mL of dimethyl sulfoxide; Preferably, 0.04 mmol of oleuropein monomer is dispersed in 2.5 mL of dimethyl sulfoxide.

3. The core-shell structured drug delivery system loaded with oleuropein according to claim 2, characterized in that: Sodium tripolyphosphate is crosslinked onto oleuropein nanoparticles in the following manner: oleuropein nanoparticles are dispersed in water, and then sodium tripolyphosphate solution is added to form a mixed solution of oleuropein nanoparticles and sodium tripolyphosphate; the pH value of the mixed solution of oleuropein nanoparticles and sodium tripolyphosphate is adjusted and stirred to obtain sodium tripolyphosphate crosslinked oleuropein nanoparticles. The mass ratio of oleuropein nanoparticles to sodium tripolyphosphate is 1:0.3-0.5; Preferably, the mass ratio of oleuropein nanoparticles to sodium tripolyphosphate is 1:0.4; Preferably, the solute mass concentration of the sodium tripolyphosphate solution is 0.8-1.8%; Preferably, oleuropein nanoparticles formed from 0.04 mmol of oleuropein monomer are dispersed in 1 mL of water.

4. The core-shell structured drug delivery system loaded with oleuropein according to claim 3, characterized in that: Folic acid was covalently linked to sodium tripolyphosphate-crosslinked oleuropein nanoparticles in the following manner: sodium tripolyphosphate-crosslinked oleuropein nanoparticles were dispersed in dimethyl sulfoxide, activated folic acid was added, and after reaction, folic acid and sodium tripolyphosphate-crosslinked oleuropein nanoparticles formed a covalent link to obtain folic acid-modified nanoparticles; the mass ratio of sodium tripolyphosphate-crosslinked oleuropein nanoparticles to folic acid was 1:0.05-0.

3. Preferably, the mass ratio of sodium tripolyphosphate cross-linked oleuropein nanoparticles to folic acid is 1:0.15; Preferably, oleuropein nanoparticles cross-linked with sodium tripolyphosphate, formed by reacting 0.04 mmol of oleuropein monomer, are dispersed in 1 mL of dimethyl sulfoxide at pH 7.

4.

5. The core-shell structured drug delivery system loaded with oleuropein according to claim 4, characterized in that: The outer shell is loaded onto the oleuropein nanoparticles in the following manner: folic acid-modified nanoparticles are dissolved in chloroform containing triethylamine, and a ferulic acid-derived lignin nanoparticle solution is added dropwise. After stirring, sonication, rotary evaporation to remove chloroform, and purification, a core-shell structured drug delivery system loaded with oleuropein is obtained. Preferably, 1-10g of folic acid-modified nanoparticles are dissolved in 0.5-2mL of chloroform containing 1-5μL of triethylamine, and 2-5mL of a 0.1-0.5% ferulic acid-derived lignin nanoparticle solution is added dropwise. Preferably, the volume of the ferulic acid-derived lignin nanoparticle solution is 3 mL, and the mass percentage of the solute is 0.3%; the folic acid-modified nanoparticles formed by reacting 0.04 mmol of oleuropein monomer are dispersed in 1 mL of chloroform containing 2.5 μL of triethylamine.

6. A core-shell structured drug delivery system loaded with oleuropein according to claim 5, characterized in that: Ferulic acid-derived lignin nanoparticles were prepared by the following method: A dimethyl sulfoxide solution of alkali lignin and a dimethyl sulfoxide solution of ferulic acid were prepared. After the ferulic acid in the dimethyl sulfoxide solution of ferulic acid was activated by the carboxyl group, the dimethyl sulfoxide solution of ferulic acid was added dropwise to the dimethyl sulfoxide solution of alkali lignin. After grafting reaction, purification, and freeze drying, ferulic acid-derived lignin powder was obtained. A dimethyl sulfoxide solution of ferulic acid-derived lignin powder was prepared. Under ice bath conditions, the dimethyl sulfoxide solution of ferulic acid-derived lignin powder was added dropwise to water while magnetic stirring was performed. After ultrasonic dispersion and purification, ferulic acid-derived lignin nanoparticles were obtained.

7. A method for preparing a core-shell structured drug delivery system loaded with oleuropein according to any one of claims 1-6, characterized in that: The following steps are performed sequentially: S1: Olive bitter glycosides were dispersed in dimethyl sulfoxide, and the resulting mixed solution was added to water. After stirring, the mixture was centrifuged and the precipitate was collected. The precipitate was purified to obtain olive bitter glycoside nanoparticles. S2: Disperse oleuropein nanoparticles in water and then add sodium tripolyphosphate solution to form a mixed solution of oleuropein nanoparticles and sodium tripolyphosphate; adjust the pH value of the mixed solution of oleuropein nanoparticles and react with stirring to obtain oleuropein nanoparticles cross-linked with sodium tripolyphosphate. S3: Disperse sodium tripolyphosphate cross-linked oleuropein nanoparticles in dimethyl sulfoxide, add activated folic acid, and after reaction, folic acid and sodium tripolyphosphate cross-linked oleuropein nanoparticles form a covalent bond to obtain folic acid modified nanoparticles. S4: Folic acid-modified nanoparticles were dissolved in chloroform containing triethylamine, and a ferulic acid-derived lignin nanoparticle solution was added dropwise. After stirring, sonication, rotary evaporation to remove chloroform, and purification, a core-shell structured drug delivery system loaded with oleuropein was obtained.

8. The method for preparing a core-shell structured drug delivery system loaded with oleuropein according to claim 7, characterized in that: In S1, 0.02-0.08 mmol of oleuropein was dispersed in 1.0-4.0 mL of dimethyl sulfoxide, and then the resulting mixed solution was added to 6-25 mL of water. After stirring at 500-1500 rpm for 0.5-2 h, the mixture was centrifuged and the precipitate was collected. The precipitate was purified to obtain oleuropein nanoparticles. In S2, the mass ratio of oleuropein nanoparticles to sodium tripolyphosphate is 1:0.3-0.5; the pH of the oleuropein nanoparticle-sodium tripolyphosphate mixed solution is adjusted to 4.5-5.5 and stirred at 200-800 rpm for 4-8 hours to obtain sodium tripolyphosphate cross-linked oleuropein nanoparticles.

9. A method for preparing a core-shell structured drug delivery system loaded with oleuropein according to claim 8, characterized in that: In S3, the ratio of sodium tripolyphosphate cross-linked oleuropein nanoparticles to folic acid is 1:0.05-0.3; In S4, 1-10 g of folic acid-modified nanoparticles were dissolved in 0.5-2 mL of chloroform containing 1-5 μL of triethylamine. 2-5 mL of a 0.1-0.5 wt% ferulic acid-derived lignin nanoparticle solution was added dropwise. After stirring in an ice bath at 8000-12000 rpm for 3-10 min, sonicating at 100-500 W for 1-5 min, removing chloroform by rotary evaporation, and purification, a core-shell structured drug delivery system loaded with oleuropein was obtained.

10. The use of a core-shell structured drug delivery system loaded with oleuropein according to any one of claims 1-6 in the preparation of a medicament for treating sepsis, characterized in that: It is used to improve the pathological damage to intestinal tissues caused by sepsis.

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